Infections are caused by a variety of microorganisms. Infections which are persistent have a myriad of consequences for the health care community including increased treatment burden and cost, and for the patient in terms of more invasive treatment paradigms and potential for serious illness or even death. It would be beneficial if an improved treatment paradigm were available to provide prophylactic treatment to prevent susceptible patients from acquiring infections as well as increasing the rate or effectiveness of eradicating the infections in patients already infected with the microorganisms.
In particular, cystic fibrosis (CF) is one example of a disease in which patients often acquire persistent or tenacious respiratory tract infections, including P. aeruginosa (PA). Another disease which is associated with recurring PA lung infections is non-CF bronchiectasis. A subset of COPD patients also suffers from PA lung infections and many have bronchiectasis.
High rates of colonization and the challenge of managing PA infections in patients with cystic fibrosis (CF) have necessitated a search for safe and effective antibiotics. Currently, inhaled tobramycin, colistin, or aztreonam is the standard of care in CF. Nothing is currently approved for treatment of patients with NTM infections, or for non-CF bronchiectasis patients.
While azithromycin possesses activity against Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pneumoniae, it has no direct activity against Pseudomonas aeruginosa, Burkholderia cepacia, or other gram-negative non-fermenters (Lode H et al., 1996). Tobramycin possesses activity against P. aeruginosa; however, the increase in the number of patients with resistant isolates on continuous therapy from ˜10% to 80% after 3 months (Smith A L et al., 1989) has led to the intermittent dosing regimen of 28-days-on followed by 28-days-off therapy. The development of a therapeutic regimen that delivers the anti-infective therapy in a continuous fashion, while still inhibiting the emergence of resistant isolates, may provide an improved treatment paradigm. It is noteworthy that chronic PA airway infections remain the primary cause of morbidity and mortality in CF patients. When patients experience pulmonary exacerbations, the use of systemic antipseudomonal therapy, frequently consisting of a β-lactam and an aminoglycoside, may result in clinical improvement and a decrease in bacterial burden. Eradication of the infection, however, is quite rare.
In CF airways, PA initially has a non-mucoid phenotype, but ultimately produces mucoid exopolysaccharide and organizes into a biofilm, which indicates the airway infection has progressed from acute to chronic. Bacteria in biofilms are very slow growing due to an anaerobic environment and are inherently resistant to antimicrobial agents, since sessile cells are much less susceptible than cells growing planktonically. It has been reported that biofilm cells are at least 500 times more resistant to antibacterial agents (Costerton J W et al., 1995). Thus, the transition to the mucoid phenotype and production of a biofilm contribute to the persistence of PA in CF patients with chronic infection by protecting the bacteria from host defenses and interfering with the delivery of antibiotics to the bacterial cell. Although much effort has been made to improve the care and treatment of individuals with CF, and the average lifespan has increased, the median age of survival for people with CF is only to the late 30s (CF Foundation web site, 2006).
Pulmonary infections from non-tuberculous mycobacteria (NTM) are also notoriously difficult to treat. They exist in the lungs in various forms, including within macrophages and in biofilms. These locations are particularly difficult to access with antibiotics. Furthermore, the NTM may be either in a dormant (termed sessile), or a replicating phase, and an effective antibiotic treatment would target both phases.
Lung infection from Mycobacterium avium subsp hominissuis (hereafter referred as M. avium) and Mycobacterium abscessus is a significant health care issue and there are major limitations with current therapies. The incidence of pulmonary infections by non-TB mycobacteria (NTM) is increasing (Adjemian et al., 2012; Prevots et al, 2010), specifically with M. avium and M. abscessus (Inderlied et al, 1993). About 80% of NTM in US is associated with M. avium (Adjemian et al., 2012; Prevots et al, 2010). M. abscessus, which is amongst the most virulent types, ranks second in incidence (Prevots et al, 2010). Diseases caused by both mycobacteria are common in patients with chronic lung conditions, e.g., emphysema, cystic fibrosis, and bronchiectasis (Yeager and Raleigh, 1973). They may also give rise to severe respiratory diseases, e.g., bronchiectasis (Fowler et al, 2006). The infections are from environmental sources and cause progressive compromising of the lung.
Current therapy often fails on efficacy or is associated with significant side-effects. M. avium infection is usually treated with systemic therapy with a macrolide (clarithromycin) or an azalide (azithromycin) in combination with ethambutol and amikacin. Oral or IV quinolones, such as ciprofloxacin and moxifloxacin, can be used in association with other compounds (Yeager and Raleigh, 1973), but higher intracellular drug levels need to be achieved for maximal efficacy. Oral ciprofloxacin has clinical efficacy against M. avium only when administered in combination with a macrolide or an aminoglycoside (Shafran et al 1996; de Lalla et al, 1992; Chiu et al, 1990). Studies in vitro and in mouse suggest that the limited activity of oral ciprofloxacin alone is related to the inability of ciprofloxacin to achieve bactericidal concentrations at the site of infection (Inderlied et al, 1989); the minimum inhibitory concentration (MIC) of 5 μg/ml versus the clinical serum Cmax of 4 μg/ml explains the limited efficacy in experimental models and in humans (Inderlied et al, 1989). M. abscessus is often resistant to clarithromycin. IV aminoglycosides or imipenem need to be applied, which often are the only available therapeutic alternatives, and these carry the potential for serious side-effects, as well as the trauma and cost associated with IV administration. Clofazimine, linezolid, and cefoxitin are also sometimes prescribed, but toxicity and/or the need for IV administration limit the use of these compounds. Thus, the available therapies have significant deficiencies and improved approaches are needed.
Recent studies also showed that both M. avium and M. abscessus infections are associated with significant biofilm formation (Bermudez et al, 2008; Carter et al, 2003): deletion of biofilm-associated genes in M. avium had impact on the ability of the bacterium to form biofilm and to cause pulmonary infection in an experimental animal model (Yamazaki et al, 2006).
Deliberate release of microbial agents in the form of mists or aerosols poses a serious bioterrorism threat. More effective methods for prevention and treatment of bioterrorism infections, particularly those that can be transmitted by inhalation, such as anthrax, tularemia, pneumonic plague, melioidosis and Q-fever, are desirable. Their stock piling in the form of frozen formulations that could be thawed to form medicines with desirable properties would be particularly attractive.
Thus, a continuing need exists for improved formulations of anti-infectives, especially for administration by inhalation. The present invention addresses this need.
Ciprofloxacin is a fluoroquinolone antibiotic that is indicated for the treatment of lower respiratory tract infections due to PA, which is common in patients with cystic fibrosis. Ciprofloxacin is broad spectrum and, in addition to PA, is active against several other types of gram-negative and gram-positive bacteria. It acts by inhibition of topoisomerase II (DNA gyrase) and topoisomerase IV, which are enzymes required for bacterial replication, transcription, repair, and recombination. This mechanism of action is different from that for penicillins, cephalosporins, aminoglycosides, macrolides, and tetracyclines, and therefore bacteria resistant to these classes of drugs may be susceptible to ciprofloxacin. Thus, CF patients who have developed resistance to the aminoglycoside tobramycin can likely still be treated with ciprofloxacin. There is no known cross-resistance between ciprofloxacin and other classes of antimicrobials.
Despite its attractive antimicrobial properties, ciprofloxacin does produce bothersome side effects, such as gastrointestinal tract (GIT) intolerance (vomiting, diarrhea, abdominal discomfort), as well as dizziness, insomnia, irritability and increased levels of anxiety. There is a clear need for improved treatment regimes that can be used chronically, without resulting in these debilitating side effects.
Delivering ciprofloxacin as an inhaled aerosol has the potential to address these concerns by compartmentalizing the delivery and action of the drug in the respiratory tract, which is the primary site of infection.
Currently there is no aerosolized form of ciprofloxacin with regulatory approval for human use, capable of targeting antibiotic delivery direct to the area of primary infection in the respiratory tract. In part this is because the poor solubility and bitterness of the drug have inhibited development of a formulation suitable for inhalation (Barker et al, 2000). Furthermore, the tissue distribution of ciprofloxacin is so rapid that the drug residence time in the lung is too short to provide additional therapeutic benefit over drug administered by oral or IV routes (Bergogne-Bérézin E, 1993).
The therapeutic properties of many drugs are improved by incorporation into liposomes. Phospholipid vehicles as drug delivery systems were rediscovered as “liposomes” in 1965 (Bangham et al., 1965). The general term “liposome” covers a variety of structures, but all consist of one or more lipid bilayers enclosing an aqueous space in which hydrophilic drugs, such as ciprofloxacin, can be encapsulated. Liposome encapsulation improves biopharmaceutical characteristics through a number of mechanisms including altered drug pharmacokinetics and biodistribution, sustained drug release from the carrier, enhanced delivery to disease sites, and protection of the active drug species from degradation. Liposome formulations of the anticancer agents doxorubicin (Myocet®/Evacet®, Doxyl®/Caelyx®), daunorubicin (DaunoXome®) the anti-fungal agent amphotericin B (Abelcet®, AmBisome®, Amphotec®) and a benzoporphyrin (Visudyne®) are examples of successful products introduced into the US, European and Japanese markets over the last two decades. Recently a liposomal formulation of vincristine (Marqibo®) was approved for an oncology indication. The proven safety and efficacy of lipid-based carriers make them attractive candidates for the formulation of pharmaceuticals.
Delivery of liposome formulations by inhalation offers many attractive features, providing that the liposome formulation is stable to the aerosolization process (Niven and Schreier, 1990; Cipolla et al, 2013). Therefore, in comparison to the current ciprofloxacin formulations, a liposomal ciprofloxacin aerosol formulation should offer several benefits: 1) higher drug concentrations, 2) increased drug residence time via sustained release at the site of infection, 3) decreased side effects, 4) increased palatability, 5) better penetration into the bacteria, and 6) better penetration into the cells infected by bacteria. It has previously been shown that inhalation of liposome-encapsulated fluoroquinolone antibiotics may be effective in treatment of lung infections. In a mouse model of F. tularensis liposomal encapsulated fluoroquinolone antibiotics were shown to be superior to the free or unencapsulated fluoroquinolone by increasing survival (CA2,215,716, CA2,174,803, and CA2,101,241).
U.S. Pat. Nos. 8,071,127, 8,119,156, 8,268,347 and 8,414,915 describe an aerosol consisting of inhaled droplets or particles. The droplets or particles comprise a free drug (e.g., an anti-infective compound) in which drug is not encapsulated and which may be ciprofloxacin. The particles further comprise liposomes which encapsulate a drug such as an anti-infective compound which also may be ciprofloxin. The free and liposome encapsulated drug are included within a pharmaceutically acceptable excipient which is formulated for aerosolized delivery. The particles may further include an additional therapeutic agent which may be free and/or in liposomes and which can be any pharmaceutically active drug which is different from the first drug. The liposomes in these patents are unilamellar vesicles (average particle size 75-120 nm). Ciprofloxacin is released slowly from the liposomes with a half-life of about 10 hours in the lung (Bruinenberg et al, 2010 b), which allows for once-a-day dosing.
Further, studies with a variety of liposome compositions in in vitro and murine infection models showed that liposomal ciprofloxacin is effective against several intracellular pathogens, including M. avium. Inhaled liposomal ciprofloxacin is also effective in treating Pseudomonas aeruginosa (PA) lung infections in patients (Bilton et al, 2009 a, b, 2010, 2011; Bruinenberg et al, 2008, 2009, 2010 a, b, c, d, 2011; Serisier et al, 2013). Compared to approved doses of oral and IV ciprofloxacin, liposomal ciprofloxacin formulations delivered by inhalation into the airways achieve much greater concentrations in the respiratory tract mucosa and within macrophages with resulting improvement of clinical efficacy: 2 hours post-inhalation of a therapeutic dose of such liposomal ciprofloxacin in patients, the concentration of ciprofloxacin in the sputum exceeded 200 μg/ml, and even 20 hours later (2 hours prior to the next dose), the concentration was >20 μg/ml, well above the minimum inhibitory concentration above for resistant mycobacteria (breakpoint of ˜4 μg/ml (Bruinenberg 2010b). Since the liposomes containing ciprofloxacin are avidly ingested by macrophages, the ciprofloxacin is brought into close proximity to the intracellular pathogens, thus further increasing anti-mycobacterial concentration and thus should lead to improved efficacy of the inhaled liposomal formulation compared to other forms of ciprofloxacin. We therefore believe that even highly resistant NTM may be suppressed with such inhaled liposomal ciprofloxacin formulations. This is significant because M. avium and M. abscessus resistance to antibiotics is common due to long-term use of systemic antibiotics in these patients. The clinical experience with PA also shows that there is no apparent emergence of resistance following inhaled liposomal ciprofloxacin therapy: in fact, even those patients who also had resistant strains initially, responded well to therapy. This is likely due to the presence of sustained overwhelming concentrations of ciprofloxacin. Furthermore, the experience with other anti-pseudomonal drugs tobramycin and colistimethate in cystic fibrosis is that even patients with resistant strains of PA respond clinically well to the inhaled form of the drugs (Fiel, 2008).
A few in vitro studies have demonstrated that liposomal ciprofloxacin is efficacious against intracellular pathogens: M. avium infection: 1) In human peripheral blood monocytes/macrophages, liposomal ciprofloxacin tested over concentrations from 0.1 to 5 μg/ml caused concentration-related reductions in intracellular M. avium-M. intracellulare complex (MAC) colony forming units (CFU) compared to free drug at the same concentrations (Majumdar et al, 1992); 2) In a murine macrophage-like cell line J774, liposomal ciprofloxacin decreased the levels of cell associated M. avium up to 43-fold and these reductions were greater than for free ciprofloxacin (Oh et al, 1995).
Once M. avium or M. abscessus infect monocytes/macrophages, the infection can then spread to the lungs, liver, spleen, lymph nodes, bone marrow, and blood. There are no published studies on the efficacy of liposomal ciprofloxacin against M. avium or M. abscessus in animal models.
Several in vivo studies have demonstrated that liposomal ciprofloxacin is efficacious against the intracellular pathogen, F. tularensis: Efficacy of liposomal ciprofloxacin delivered to the lungs by inhalation or intranasal instillation against inhalational tularemia (F. tularensis LVS and SCHU S4) in mice, was demonstrated with as little as a single dose of liposomal ciprofloxacin providing 100% protection post-exposure, and even effective post-exposure treatment for animals that already had significant systemic infection (Blanchard et al, 2006; Di Ninno et al, 1993; Conley et al, 1997; Hamblin et al, 2011; Wong et al, 1996). The studies also found that inhaled liposomal ciprofloxacin was superior to both inhaled and oral unencapsulated ciprofloxacin.
In contrast, a) free ciprofloxacin was inferior to liposomal ciprofloxacin in macrophage models of mycobacterial infections (Majumdar et al, 1992; Oh et al, 1995); b) free ciprofloxacin alone delivered to the lungs had inferior efficacy to free ciprofloxacin when tested in murine models of F. tularensis infection (Conley et al, 1997; Wong et al, 1996), as it is rapidly absorbed into the blood stream. A formulation made up of both free and liposomal ciprofloxacin combines the potential advantages of an initial transient high concentration of free ciprofloxacin to increase Cmax in the lungs, followed by the slow release of ciprofloxacin from the liposomal component, as demonstrated in BE (Cipolla et al, 2011; Serisier et al, 2013). The free ciprofloxacin component also has a desirable immunomodulatory effect (U.S. Pat. Nos. 8,071,127, 8,119,156, 8,268,347 and 8,414,915).
Further, liposomal ciprofloxacin injected parenterally activates macrophages, resulting in increased phagocytosis, nitric oxide production, and intracellular microbial killing even at sub-inhibitory concentrations, perhaps via immunostimulatory effects (Wong et al, 2000). The ciprofloxacin-loaded macrophages may migrate from the lungs into the lymphatics to treat infections in the liver, spleen, and bone marrow—as suggested by the systemic effects of pulmonary-delivered CFI in tularemia (Di Ninno et al, 1993; Conley et al, 1997; Hamblin et al, 2011, Wong et al, 1996). Liposome-encapsulated antibiotics are also known to better penetrate bacterial films in the lungs (Meers et al, 2008). The anti-mycobacterial and immunomodulatory effects of the new formulations delivered to the lungs, may therefore provide a better alternative to the existing treatments for patients infected with M. avium or M. abscessus, or provide an adjunct for incremental improvements.
A pharmacokinetic study of liposomal ciprofloxacin demonstrated high uptake by alveolar macrophages in animals, which is presumably the reason for the highly effective post-exposure prophylaxis and treatment of inhalational tularemia in mice. Although the plasma levels of ciprofloxacin were low following respiratory tract administration of the liposomal ciprofloxacin, a reduction of the tularemia infection from the liver, spleen, tracheobronchial lymph nodes, as well as the lungs, was observed suggesting that the alveolar macrophages loaded with liposomal ciprofloxacin migrate from the lungs via lymph into the liver, spleen and lymph nodes (Conley et al, 1997).
It would be valuable to be able to prolong the shelf life of liposomally encapsulated antibiotics. However, such formulations, such as liposomal ciprofloxacin formulations, are notoriously sensitive to freeze-thaw. For example, after freeze-thaw of the liposomal ciprofloxacin formulations described above, agglomerates of lipids are observed indicating that many of the liposomes do not retain their integrity in response to the stress of freeze-thaw. These thawed formulations certainly could not be effectively used, e.g., as aerosolized due to the physical agglomerates.
It would be ideal to identify a liposome formulation that retains its stability and integrity after freeze-thaw. A frozen formulation would have a longer shelf-life than a refrigerated or room-temperature formulation due to the reduction in mobility of water and the other constituents resulting in a reduction in the rate of the degradation processes (e.g., lipid hydrolysis). There has been extensive literature describing the challenges of freezing liposomes and maintaining liposome integrity following freeze-thaw. Cryoprotectants such as dimethylsulfoxide, glycerol, quaternary amines and carbohydrates have shown promise (Wolkers et al., 2004). It is also well-established that sugars can stabilize phospholipid vesicles during freezing and this stabilization requires direct interaction between sugar and the phospholipid head group (Strauss et al, 1986; Crowe et al, 1988; Izutsu et al, 2011; Stark et al, 2010, Siow et al, 2007; Siow et al, 2008). The addition of sugar, e.g. polyols, to both the internal liposomal fluid and extraliposomal fluid can improve the robustness of liposomes to freeze-thaw and help to maintain liposome integrity. However, not all liposome formulations are fully protected by sugars and in many cases there will be a proportion of vesicles which lose their integrity completely, and others which agglomerate leading to an increase in vesicle size. These events are also associated with loss of encapsulated drug (Strauss et al, 1986; Crowe et al, 1988; Izutsu et al, 2011; Stark et al, 2010, Siow et al, 2007; Siow et al, 2008).
The ability to modify beneficially the properties of the liposome formulation following freeze-thaw has also not been anticipated. Certainly, it is most likely to degrade the liposomes following freeze-thaw, such that the integrity of the liposomes is compromised. However, there have been no published reports of retention of liposome integrity following freeze thaw while simultaneously modifying the drug encapsulation and drug release properties in a beneficial way.
In addition, there have been no reported examples of liposomes containing drug nanocrystals following freeze-thaw. The presence of drug in the form of nanocrystals within the liposomes would have the potential to alter the release properties of the drug, as there are now two factors or constraints affecting the rate of release; i.e., the liposome membrane is one barrier and the requirement for dissolution of the drug from the crystal form prior to transport through the lipid bilayer is the second. Modifying the size and shape of the crystals in the liposomes will allow the release rate to be further adjusted. The size and shape of the crystals can be adjusted by changing the proportions of excipients in the formulation, i.e., increasing or decreasing the concentration of the drug, liposomal lipids, cryopreservative and surfactant. The presence of drug nanocrystals within the liposomes has the potential to improve other properties of the formulation, including its stability characteristics. These modifications in total may improve the therapeutic effect of the liposome formulation or allow for greater convenience in administration profile; e.g., a reduction in the frequency of administration. The improved administration profile could lead to greater patient compliance and thus increased efficacy. The absence of peaks of drug concentration due to slower dissolution and release could also reduce or eliminate undesirable adverse effects with drug crystals that dissolve slowly.
Another opportunity is to create an immediate release profile that is combined with the sustained release profile. After thawing the formulation there may be a proportion of drug which is released from the liposomes and so becomes immediately available upon inhalation. This proportion of “free drug” can be adjusted to between 1 and 60%, or 10 and 50%, or 20 to 40% by adjusting the proportions of excipients in the formulation, i.e., increasing or decreasing the concentration of cryopreservative and/or surfactant. The cryopreservatives may include polyols, sugars, including sucrose, trehalose, lactose, mannital, etc. Surfactants may include non-ionic surfactants including the polysorbates such as polysorbate 20 (also called tween 20). The cryopreservatives may be present either on the inside (intraliposomally) of the liposomes, and on the outside of the liposomes (extraliposomally), or both.
There have been a number of liposomal formulations that contain drug in a precipitated, gel or crystalline form within the liposomes, but all of these drug precipitates are created during the initial drug loading process. For example there are reports of crystallized doxorubicin (Lasic et al, 1992; Lasic et al, 1995; Li et al, 1998), topotecan (Abraham et al, 2004) and vinorelbine (Zhigaltsev et al. 2006) in liposomes after ion/pH gradient loading (Drummond et al, 2008). There have been no reports of liposomes containing encapsulated drug wherein some of the drug forms drug crystals following freeze-thaw.